A polypeptide with self-assembly and adhesion properties and its application

Abstract

The present invention discloses a polypeptide with self-assembly and adhesion properties and its application, wherein the polypeptide is from Lactobacillus acidophilus ( Lactobacillus acidophilus )CICC 6074, the amino acid sequence is shown as ATTINASSSAINTNTNAKYDVDVT. The polypeptide of the present invention can self-assemble on the polar liposomes, thereby maintaining the stability of the polar liposomes; the polypeptide-modified polar liposomes can adhere to the surface of HT-29 cells, which can be used to improve the intestinal adhesion of the polar liposomes and promote efficient drug absorption. Studies have shown that the polypeptide of the present invention can be used as a delivery material for nano-drugs, and can be used in the fields of food, medicine, etc., and has a good market prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a polypeptide with self-assembly and adhesion properties and applications thereof. Background Art

[0002] Lactic acid bacteria are a common probiotic in humans and animals, and have many beneficial effects on the human body. They can grow and reproduce in the digestive tract to inhibit the growth and infection of harmful bacteria and antagonize pathogens, increase intestinal nonspecific immunity, improve human immunity, and prevent diseases, so they are widely used. Among them, Lactobacillus acidophilus is an important probiotic that has been approved for use in health food, and has the characteristics of bile salt resistance, acid resistance, safety and non-toxicity. Among them, Lactobacillus acidophilus CICC 6074 can adhere to the small intestinal epithelial cells, reduce the apoptosis of the small intestinal epithelial cells, and thus prevent necrotizing enterocolitis; at the same time, Lactobacillus acidophilus can also adhere to the surface of HT-29 cells, induce HT-29 cell apoptosis and prevent cell invasion.

[0003] Liposomes are nano-spherical vesicles, which are composed of an inner aqueous phase and one or more phospholipid bilayers encapsulating the inner aqueous phase, and have a cell-like structure. Liposomes can exert a variety of functional properties in the body, and have received widespread attention. They are currently often used as drug carriers. When liposomes enter the body, they are mainly engulfed by the reticuloendothelial system and activate the body's autoimmune function. At the same time, liposomes can also change the absorption and distribution of encapsulated drugs in the body, causing the drugs to accumulate in tissues and organs such as the heart, liver, lungs, and bone marrow, thereby reducing the therapeutic dose of the drug, reducing the toxicity of the drug, and achieving the purpose of targeted drug delivery. Liposomes can be degraded in the body, are non-toxic, and have no immunogenic properties, and are excellent materials for use as drug carriers. Therefore, the research on liposomes as drug carriers has received increasing attention.

[0004] However, ordinary liposomes currently have disadvantages such as passive targeting, low targeting, unsatisfactory treatment of certain diseases, and poor stability, so liposomes need to be modified. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a polypeptide with adhesion and self-assembly properties and its application. The surface of the emulsion liposome is modified by the polypeptide with self-assembly and adhesion properties. Not only does the modified emulsion liposome have adhesion and stronger targeting, ensuring that the drug is fully released at the lesion site, but the stability of the emulsion liposome is also improved. The modification process is also automatic, efficient, safe and non-toxic.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A polypeptide with self-assembly and adhesion properties, the polypeptide is from Lactobacillus acidophilus CICC 6074, and the amino acid sequence is shown in SEQ ID NO.1. The polypeptide has the ability to adhere to cells and realize self-assembly on milk polar liposomes.

[0008] Wherein, SEQ ID NO.1:

[0009] Ala-Thr-Thr-lle-Asn-Ala-Ser-Ser-Ser-Ala-lle-Asn-Thr-Asn-Thr-Asn-Ala-Lys-Tyr-Asp-Val-Asp-Val-Thr.

[0010] As an improvement, the cells are HT-29 cell surfaces.

[0011] A kind of emulsion polar liposome modified by the polypeptide with self-assembly and adhesion properties, the surface of the liposome is modified to form a protective film outside the emulsion polar liposome, and the modified liposome has better stability when delivering drugs.

[0012] The application of the above-mentioned polypeptide-modified emulsion polar liposomes with self-assembly and adhesion properties in the preparation of targeted drugs.

[0013] A targeted drug is delivered through the above-mentioned polypeptide-modified milk polar liposome with self-assembly and adhesion properties.

[0014] Beneficial effects:

[0015] Compared with the prior art, the present invention provides a polypeptide with self-assembly and adhesion properties and its application. The polypeptide of the present invention has self-assembly and adhesion properties, which is the first report. At the same time, it can be used as an excellent delivery material in the fields of medicine, food, etc., and has broad market prospects. Specifically:

[0016] 1. The polypeptide of the present invention can complete self-assembly on the surface of liposomes, and can be used to modify the emulsion liposomes, protect the emulsion liposomes from external interference, and help improve the stability of the emulsion liposomes;

[0017] 2. The cell adhesion function of the polypeptide of the present invention can be used to improve the intestinal adhesion of the delivery material of nano-drugs, which is beneficial to promote drug absorption. The polypeptide is safe, efficient, and has no toxic side effects, and can be widely used in various fields such as food and medicine;

[0018] 3. The emulsion liposomes modified by the polypeptide of the present invention also have the ability to adhere to the surface of the corresponding cells, and can carry nano-drugs and fix them on the cells on the surface of the corresponding target organs, so that the drugs can play a role in specific organs and at the same time prolong the duration of drug action. That is, the polypeptide modification of liposomes can not only improve the stability of the emulsion liposomes, but also improve the targeting of the emulsion liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the assembly diagram of different polypeptide fragments and emulsion liposomes;

[0020] Figure 2 The figure shows the adhesion of different polypeptide fragments to HT-29 cells. DETAILED DESCRIPTION

[0021] Below by specific embodiment, and according to data and experimental result, the present invention is further described.These embodiments are for illustrating the present invention, but not limit the scope of the present invention in any way.Unless otherwise stated, generally have the meaning that those of ordinary skill in the art are usually understood.

[0022] Example 1 Self-assembly ability of polypeptide sequences

[0023] Lactobacillus acidophilus CICC 6074 was purchased from China Industrial Microbiological Culture Collection Center.

[0024] When proteomic analysis of Lactobacillus acidophilus CICC 6074 was performed, peptides N1, N2, N3, N4, and N5 were found to be located on the surface of lactic acid bacteria, among which the amino acid sequences of the five peptides are shown in Table 1. The peptides were synthesized by Shanghai Qiangyao Biological Co., Ltd. and labeled with FITC.

[0025] Table 1 Amino acid sequences of different polypeptide fragments

[0026]

[0027] 2. Preparation of polar liposomes

[0028] The milk polar liposomes were prepared by thin film ultrasonic dispersion method, referring to the method of Yang, Y, etc. and modified. 30 mg milk fat globule phospholipids, 10 mg cholesterol and 3 mg hexadecylamine were mixed and dissolved in 4 mL of chloroform, and the mixture was transferred to a rotary evaporator, evaporated in a 50 ° C water bath under a vacuum environment of -0.01 kPa until a thin film appeared in the round-bottom flask, and nitrogen was introduced to wash away the residual chloroform. Then 4 mL of PBS solution (pH 7.0) was added to the flask, hydrated for 1 h under an environment above 50 ° C, and then dissolved in PBS solution. After the dispersion was treated with ice water bath ultrasound, the ultrasound treatment was carried out at a frequency of 130w, running for 5s, stopping for 7s, a total of 50 cycles, and a total treatment of 10min. The suspension was treated with ultrasound until it was uniform and transparent, and passed through a 0.45 μm filter membrane to obtain milk polar liposomes, which were stored in a refrigerator at 4 ° C for later use.

[0029] 3. Self-assembly of polypeptide fragments on milk polar liposomes Add 20 μL of lissamine rhodamine B solution (1 mg / mL) to 1 mL of milk polar liposome solution (1 mg / mL), shake thoroughly at 25°C for 15 min; centrifuge the solution after the reaction for 30 min (4°C, 12000 rpm), collect the lower precipitate, and wash it three times with pH 7.0 PBS buffer, and suspend the liposomes in PBS buffer at a concentration of 1 mg / mL. Add the polypeptide fragment (200 μg / mL) to an equal volume of milk polar liposomes at 25°C and shake at 50 rpm for 2.5 h. Centrifuge the mixed solution, wash the precipitate with PBS for 3 times, and use a confocal laser microscope to observe the self-assembly of the polypeptide fragment on the milk polar liposome under a 100x oil microscope.

[0030] 4. Results Analysis

[0031] like Figure 1 As shown, under a microscope, it can be observed that the red liposomes labeled with lissamine rhodamine B are surrounded by green FITC-labeled N1, N2, N3, N4, and N5. The specific self-assembly description is shown in Table 2. As can be seen from Table 2, the five polypeptide fragments synthesized in Example 1 all have the ability to self-assemble.

[0032] Table 2 Self-assembly of polypeptide fragments

[0033]

[0034] Example 2 Adhesion ability of polypeptide fragments

[0035] 1. Culture of HT-29 cells

[0036] HT-29 cells were cultured in complete culture medium (DMEM medium containing sodium pyruvate: fetal bovine serum: double antibody = 100:10:1) under the following culture conditions: temperature of 37°C, 95% air, 5% CO2, and humidity of 70%-80%. The selected HT-29 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0037] After 2-3 cell passages, when the percentage of adherent cells reached about 80%, HT-29 cells were plated at 4-5×10 5 cells / cm 2 The cells were seeded at a density of 1.54 μg / mL on a 24-well plate. After 24 h of culture, the cells proliferated and differentiated into a monolayer of cells.

[0038] 2. Adhesion of peptide fragments to HT-29 cells

[0039] Peptide N1, peptide N2, peptide N3, peptide N4, and peptide N5 were dissolved in PBS buffer solution (pH 7.4) to a concentration of 3 μg / mL. The chamber was washed with PBS buffer (pH 7.4), and the peptide fragment solution was added to the chamber. After incubation at 37°C for 2 hours, it was rinsed three times with PBS buffer, and the adhesion of different peptide fragments to HT-29 cells was observed using a TH4-200 inverted fluorescence microscope.

[0040] 3. Results Analysis

[0041] The results are as follows Figure 2 As shown, when the polypeptide fragments were incubated with HT-29 cells, the fluorescence density of N1 was significantly higher than that of N2, N3, N4, and N5. The adhesion conditions are shown in Table 3. The results show that N1 can adhere to HT-29 cells.

[0042] Table 3 Adhesion of peptide fragments

[0043]

[0044] In summary, the peptides extracted from L.acidophilus CICC 6074 have the ability to self-assemble, and the N1 peptide has both self-assembly and good adhesion. This segment of peptide has self-assembly and adhesion properties. The emulsion liposomes modified with this peptide can be used to deliver nano-drugs, which is beneficial to improve the stability of the nano-delivery system and increase the intestinal adhesion of nano-materials.

Claims

1. A milky polar liposome modified with a polypeptide having self-assembly and adhesion properties, characterized in that: The polypeptide is from Lactobacillus acidophilus CICC 6074, and the amino acid sequence is shown in SEQ ID NO.

1. The polypeptide has the function of adhering to cells and realizes self-assembly on milk polar liposomes.

2. The use of the polypeptide-modified emulsion polar liposome with self-assembly and adhesion properties according to claim 1 in the preparation of targeted drugs, characterized in that: The targeted drug adheres to the surface of the intestinal cancer cell HT-29.

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